Isolation and characterization of a Halomonas species for non-axenic growth-associated production of bio-polyesters from sustainable feedstocks

ABSTRACT Biodegradable plastics are urgently needed to replace petroleum-derived polymeric materials and prevent their accumulation in the environment. To this end, we isolated and characterized a halophilic and alkaliphilic bacterium from the Great Salt Lake in Utah. The isolate was identified as a Halomonas species and designated “CUBES01.” Full-genome sequencing and genomic reconstruction revealed the unique genetic traits and metabolic capabilities of the strain, including the common polyhydroxyalkanoate (PHA) biosynthesis pathway. Fluorescence staining identified intracellular polyester granules that accumulated predominantly during the strain’s exponential growth, a feature rarely found among natural PHA producers. CUBES01 was found to metabolize a range of renewable carbon feedstocks, including glucosamine and acetyl-glucosamine, as well as sucrose, glucose, fructose, and further glycerol, propionate, and acetate. Depending on the substrate, the strain accumulated up to ~60% of its biomass (dry wt/wt) in poly(3-hydroxybutyrate), while reaching a doubling time of 1.7 h at 30°C and an optimum osmolarity of 1 M sodium chloride and a pH of 8.8. The physiological preferences of the strain may not only enable long-term aseptic cultivation but also facilitate the release of intracellular products through osmolysis. The development of a minimal medium also allowed the estimation of maximum polyhydroxybutyrate production rates, which were projected to exceed 5 g/h. Finally, also, the genetic tractability of the strain was assessed in conjugation experiments: two orthogonal plasmid vectors were stable in the heterologous host, thereby opening the possibility of genetic engineering through the introduction of foreign genes. IMPORTANCE The urgent need for renewable replacements for synthetic materials may be addressed through microbial biotechnology. To simplify the large-scale implementation of such bio-processes, robust cell factories that can utilize sustainable and widely available feedstocks are pivotal. To this end, non-axenic growth-associated production could reduce operational costs and enhance biomass productivity, thereby improving commercial competitiveness. Another major cost factor is downstream processing, especially in the case of intracellular products, such as bio-polyesters. Simplified cell-lysis strategies could also further improve economic viability.

Values for salinity, pH, and temperature were obtained when cultivating the strain on Nutrient Broth (with the respective other parameters optimized).Specific growth rates, as well as PHB production yields and -rates, were obtained when cultivating CUBES01 on chemically-defined (minimal) medium, complemented with the indicated substrate.The expected sizes of amplicons were 0.9 kbp and 5.1 kbp for pBBR1MS, and 2.9 kbp and 6.5 kbp for pTJS140.Combinations of primers correspond to full sequence coverage of each plasmid, confirming that the vectors were, in fact, maintained and intact.

FIG 1 FIG 2 FIG 3 FIG 4
FIG 1 Codon Usage Bias (CUB) of Halomonas sp.CUBES01, based on the annotated open-reading frames differentiated by contig 1 and contig 2. The ∆ is the absolute percentage difference between the two individual contigs.

FIG 5
FIG 5 NMR and GPC analyses of polyester obtained from Halomonas sp.CUBES01 when grown on a semi-minimal medium (modified MM-G as per Chen et al., 2017 [11]) with acetate as the primary substrate.(a) Nuclear Magnetic Resonance spectrum for protons ( 1 H-NMR) of the obtained bio-polyester.The poly(hydroxyalkanoate) was composed of 3-hydroxybutyrate repeat-units, according to the sextet resonance at a chemical shift of 5.25 ppm.(b) Elugram of Gel-Permeation Chromatography with the bio-polyester.

FIG 6 FIG 7
FIG 6 Microscopy of Halomonas sp.CUBES01 cells stained with Nile red.The samples were obtained from late exponential growth-phase, during cultivation on chemically-defined medium with sucrose (a) and glycerol (b), corresponding to FIG 4. Additional microscopy images are provided in SI3.

FIG 8 Gel
FIG 8 Gel Electrophoresis of amplicons from confirmation PCR of Halomonas sp.CUBES01 mutants bearing the plasmids pBBR1MCS (top) or pTJS140 (bottom).The expected sizes of amplicons were 0.9 kbp and 5.1 kbp for pBBR1MS, and 2.9 kbp and 6.5 kbp for pTJS140.Combinations of primers correspond to full sequence coverage of each plasmid, confirming that the vectors were, in fact, maintained and intact.

FIG 9
FIG 9 Osmolysis test with Halomonas sp.CUBES01.(a) Change of optical density (OD600) over time during osmolysis of Halomonas cells in salt-free water.(b) Cell viability test (10 4 -fold dilution) on solid growth medium (Nutrient Broth with 1 M sodium chloride) by colony formation as a measure of osmolysis.Top row are agar-plates containing samples from the original culture (OD600 of 0.6), as opposed to the bottom row containing samples after exposure of the cells to deionized water for 1 minute.

TABLE 3 : Susceptibility of Halomonas sp. CUBES01 to different antibiotics
. Phenotypes of CUBES01 were determined by DSMZ as a service when grown on M1 medium with 10% sodium chloride at 30 • C for 48 hours.The diameter of inhibition zone is interpreted into no inhibition (-), 0.1-1.6 cm (+), and > 1.6 cm (++), respectively.

TABLE 5 Plasmid-vectors used in the present study
. oriV: origin of vegetative replication, Stm R : streptomycin resistance, Spm R : spectinomycin resistance, Kan R : kanamycin resistance, Neo R : neomycin resistance

TABLE 6 Primers used in the present study for
amplification of 16S rRNA and confirmation of self-replicating plasmids in Halomonas sp.CUBES01 via PCR.

TABLE 7 :
Halomonas species and Genbank IDs used in phylogenetic comparison.

TABLE 8
Correlation of biomass concentration (in optical density) to cell dry weight.